摘要
试验旨在建立一套完整的绵羊甲状腺细胞的原代培养方法,为绵羊甲状腺功能的体外研究奠定基础。通过常规消化分离得到甲状腺滤泡上皮细胞,光镜下对不同培养时间的细胞进行形态学观察,并结合RT-PCR、细胞免疫荧光和ELISA,对培养的绵羊甲状腺细胞从形态,甲状腺球蛋白(TG)、甲状腺过氧化物酶(TPO)、促甲状腺激素受体(TSHR)特异基因mRNA表达,TG特异抗原蛋白表达和甲状腺激素T3、T4的分泌功能等方面进行鉴定。结果显示,绵羊甲状腺细胞在体外呈贴壁式生长,具有上皮样细胞特点;TG染色呈胞浆阳性,具有特异TPO、TG、TSHR mRNA表达及分泌T3、T4的功能,但T3、T4的分泌量随培养时间的延长呈递减趋势。本研究成功地建立了简便可行的绵羊甲状腺细胞的原代培养方法,为深入研究绵羊甲状腺的功能提供了试验平台。
This study established a primary culture model of sheep thyroid cells, and paved the way for in vitro study on thyroid functions in sheep. In this experiment, thyroid gland was dissec- ted,digested with trypsin and collagenase Ⅰ ,purified by centrifugation and seed on 6 well plates. The cells were characterised bymorphologic observation,mRNA detection of genes specifically ex- pressed in thyroid cells, including thyroglobulin （TG）, thyriod peroxidase （TPO） and thyroid- stimulating hormone receptor （TSHR） using RT-PCR （reverse transcription-polymerase chain reaction） and specific antigen protein （TG） expression with immunofluorescence technique. Meanwhile,secretion of thyroid hormones T3 ＆ T4 was analysed using ELISA （enzyme linked immunosorbent assay） at different culture time points. The results showed that the cells could ad- hereceto plate and grew well. The cells also presented all the characteristics of the epithelioid cells,expressed thyroid specific genes and maintained the functions of secreting T3 ＆ T4 thyroid hormones, though the secretion amount of the hormones tended to decline along with the culture time. It indicated that thyroid epithelioid cells were obtained in vitro.
出处
《中国畜牧兽医》
CAS
北大核心
2015年第2期408-413,共6页
China Animal Husbandry & Veterinary Medicine
基金
教育部博导类基金(20126518110004)
关键词
绵羊
甲状腺
细胞原代培养
形态学
鉴定
sheep
thyroid gland
primary cell culture
morphology
identification
